SiRNA TARGETING ETS1 AND ELK1 AND METHOD OF USING SAME IN THE INHIBITION OF CIP2A GENE IN CANCER TREATMENT

ABSTRACT

Disclosed are methods of attenuating activity of the gene promoter of CIP2A. siRNAs are used to target against Ets1 and Elk1 transcriptional factors, thereby blocking the binding of Ets1 and Elk1 to the CIP2A gene promoter. It is disclosed that the siRNAs targeted against Ets1 and Elk1 attenuate the gene expression of CIP2A. A kit containing siRNA reagents for attenuating the CIP2A gene expression is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No. 13/776,878filed Feb. 26, 2013, which claims the benefit under 35 U.S.C. §119(e) toU.S. Provisional Application No. 61/722,386 filed Nov. 5, 2012 and U.S.Provisional Application No. 61/604,152 filed Feb. 28, 2012, the contentsof which are incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The present invention generally relates to the field of studying thepromoter activity of the CIP2A oncogene and binding of transcriptionalfactors to the CIP2A gene promoter in cancer pathogenesis. Specifically,the present invention provides the use of siRNA targeting againsttranscriptional factors Ets1 and Elk1 to reduce their binding to theCIP2A gene promoter so as to inhibit the CIP2A gene activity and therebyattenuate tumor development.

BACKGROUND OF THE INVENTION

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a 90 kDaauto-antigen reported to be expressed in hepatocellular carcinoma ofcancer patients (Hoo et al. 2002). It is believed to be an oncoprotein.CIP2A has a diverse array of functions including the inhibition ofprotein phosphatase 2A (PP2A) via dephosphorylation of c-Myc. It ishypothesized that CIP2A stabilizes c-Myc activity, probably via thedephosphorylation of c-Myc at its amino acid 62 position of serine (S62)(Junttila and Westermarck, 2008). Li et al. and Côme et al. similarlydisclose CIP2A's interaction with c-Myc. CIP2A may play a role inprogression of cancers, including human cervical, gastric, and breastcancers (Junttila et al. 2007; Li et al. 2008; Côme et al. 2009).

Increased expression of CIP2A is reported in tissue samples fromgastric, breast, oral, prostate, cervical, esophageal squamous cell, andnon-small cell lung cancer, colon, ovarian and colorectal patients(Khanna et al. 2009; Katz et al. 2010; Vaarala et al. 2010; Liu et al.2011; Qu et al. 2010; Dong et al. 2011). The increased expression ofCIP2A in breast, non-small cell lung and early stage tongue cancer isfound to be correlated with poor prognosis and therefore may serve as abiomarker for cancer disease progression (Khanna et al. 2008; Dong etal. 2011; Böckelman et al. 2011).

Considering the increased expression of CIP2A in various types of cancerand its putative role in cancer progression through inhibition of PP2Atumor suppressor activity, it is important to identify and characterizetranscription factors that regulate CIP2A gene promoter and thus CIP2Agene regulation. However, scare information exists regarding CIP2Apromoter regulation. Recently, Khanna et al. (2011) disclosed a role ofa transcriptional factor (Ets1) in transcriptional regulation of CIP2Ain human gastric and prostate cancer cells. These authors furtherspeculated that Ets1 may act via the mitogen activated protein kinasesignaling cascade. The exact role of Ets1 and other potentialtranscriptional factors requires confirmation. The transcriptionalelements regulating the expression of CIP2A still remain unclear.Furthermore, whether the proposed transcriptional regulation may beapplicable in other cancer cells is unknown.

There is a continuing need in understanding the transcriptionalregulation of CIP2A and in identifying means to suppress the expressionof CIP2A and attenuate cancer development. The present inventionprovides a novel approach to use siRNA to influence the CIP2A promoteractivity and thus altering the cancer pathogenesis in human.

SUMMARY OF THE INVENTION

The present invention provides characterization the CIP2A promoterregion and identification of specific transcription factors (i.e., Ets1and Elk1) that regulate the CIP2A promoter. It is discovered that theCIP2A promoter regulation is cell type specific. Specifically, Ets1 andElk1 are required to regulate CIP2A gene expression in cervical cancercells as well as endometrial cancer cells.

The present invention provides a method of using siRNA approachtargeting against the transcriptional factors to down regulate the CIP2Aexpression in human urogenital cancers including cervical andendometrial carcinoma cells. The present siRNA approach has practicalutility in attenuating tumor oncogenes in women's diseases.

The present invention provides siRNAs as well as compositions of siRNAfor inhibiting the expression of the CIP2A gene in a mammal. The presentinvention also provides compositions and methods for treatingpathological conditions and diseases mediated by the expression of theCIP2A gene, such as in cervical or endometrial cancer. The siRNA of thepresent invention comprises an RNA strand (the anti-sense strand) havinga region sufficient to hybridize to mRNA of CIP2A and causes CIP2A mRNAto degrade. Preferably, the siRNA is more than 15 nucleotides and lessthan 30 nucleotides in length, generally 20-25 nucleotides in length,and is substantially complementary to at least part of an mRNAtranscript of the CIP2A gene.

In one aspect, the present invention provides a method of attenuatingthe gene expression of CIP2A, comprising the steps of: a) providing afirst siRNA targeted against Ets1 transcriptional factor and a secondsiRNA targeted against Elk1 transcriptional factor; b) exposing thefirst siRNA and the second siRNA to a cell suspected of developing intotumor, wherein the first siRNA and the second siRNA attenuate the geneexpression of CIP2A, thereby attenuating the tumor development of saidcell.

In another aspect, the present invention provides a method forinhibiting CIP2A expression in a human, comprising the steps ofadministering to a human suspected of suffering from a cancer aneffective amount of a first siRNA and a second siRNA, wherein the firstsiRNA targets against Ets1 and the second siRNA targets against Elk1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic representation of putative transcriptionsites spanning the entire ˜200 bp CIP2A promoter from the transcriptionstart site (i.e., ATG). The putative transcription factor binding sitesdo not represent an exhaustive list and the transcription sites wereidentified using a bioinformatics program. The putative transcriptionfactor binding sites have the following nucleotide sequences: 1)glucocorticoid receptor alpha: (AATCA); 2) Retinoic acid receptor alpha:(ACTTCCGGAG); 3) Pax5: (GGAGCCC); 4) Ets1: (CTTCCGG) or (CCGGAAG); 5)Elk1: (CTTCCGGAG) or (ATCCGGAAG); 6) NF-_(k)β: (GGGACTTCC); 7) Sp-1:(TGGGCGGTGG); and 8) AP-2: (CGGGCCGCGG).

FIG. 2 depicts a schematic representation of the pGL4.10 [luc2] plasmid.This plasmid is 4,242 bp in length and contains the luciferase reportergene and multiple cloning sites upstream of the luc gene. Additionalfeatures of the pGL4.10 [luc2] plasmid are indicated, including the NheIand XhoI restriction sites.

FIG. 3 depicts a schematic representation of the pGL4.10 [luc2] plasmidcontaining the CIP2A promoter. This plasmid is 6,677 bp in length andcontains 2,379 bp upstream of the CIP2A coding gene region. Additionalfeatures of the CIP2A−2379/+70 are indicated, including the NheI andXhoI restriction sites.

FIG. 4 depicts the nucleotide sequence of the ˜2.5 kb CIP2A promoter(SEQ ID NO: 1). The 2,448 bp CIP2A promoter fragment was obtainedthrough PCR amplification from a genomic DNA using primers directed toamplify positions 7301-4854 on the GenBank Accession Number AC 092693.8bearing the Homo sapiens 3q BAC clone RP11-161J9 complete sequence.

FIG. 5 depicts a schematic representation of a total of nine (9) CIP2Apromoter luciferase (Luc) reporter constructs. Each of these reporterconstructs contains a CIP2A promoter fragment (˜2.5 kb to ˜0.1 kb inlength upstream of the CIP2A transcription start site (i.e., ATG)) thatis fused to with the Luc reporter gene.

FIG. 6 depicts a schematic representation of the pRL-TK plasmid. Thisplasmid is 4,045 bp in length and contains the luciferase reporter geneRenilla and multiple cloning sites upstream of the Rluc gene. The pRL-TKplasmid consists of HSV TK promoter is used for optimization oftransfection efficiency in luciferase assay.

FIG. 7 depicts a luciferase assay in human cervical carcinoma cells(HeLa), which were transfected with various CIP2A promoter constructs asshown in FIG. 4 and assayed for luciferase activity after 48 hours. Foldincrease in relative luciferase activity (RLA) was compared with pGL4basic (value is set at 1). Normalization in transfection efficiency wasperformed by co-transfection with pRL-TK (Renilla expression vector).The means±S.D. are from three different experiments, each performed intriplicate.

FIG. 8 depicts a luciferase assay in human liver carcinoma cells(HepG2), which were transfected with various CIP2A promoter constructsshown in FIG. 4 and assayed for luciferase activity after 48 hours. Foldincrease in relative luciferase activity (RLA) was compared with pGL4basic (set as 1). Normalization in transfection efficiency was performedby co-transfection with pRL-TK (Renilla expression vector). Themeans±S.D. are from three different experiments, each performed intriplicate.

FIG. 9 depicts a luciferase assay in human endometrial carcinoma cells(ECC1), which were transfected with various CIP2A promoter constructs asshown in FIG. 4 and assayed for luciferase activity after 48 hours. Foldincrease in relative luciferase activity (RLA) was compared with pGL4basic (set as 1). Normalization in transfection efficiency was performedby co-transfection with pRL-TK (Renilla expression vector). Themeans±S.D. are from three different experiments, each performed intriplicate.

FIG. 10 depicts a schematic representation of six different mutantsobtained utilizing site-directed mutagenesis. Transcription factorbinding sites mutated in the CIP2A promoter region are indicated inbold, double-strike and are underlined. CIP2A Mut1 targeted the bindingsites for transcription factor NF-κB, CIP2A Mut2 targeted the bindingsite for transcription factor Ets1, CIP2A Mut3 was targeted towards theElk1 binding site, and CIP2A Mut4 targeted the binding site fortranscription factor Pax-5, while CIP2A Mut 5 and CIP2A Mut 6 targetedthe binding sites for Ets1/Elk1. FIG. 10 also depicts the Ets1 and Elk1binding sites control transcription of CIP2A in which six (6) differentmutants were constructed. Mutated binding sites in the CIP2A promoterregion are indicated in bold and are underlined. Deletions are indicatedin bold, italics, and are underlined. CIP2A Mut1 targeted the bindingsites for transcription factor NF-κB, CIP2A Mut2 targeted the bindingsite for transcription factor Ets1, CIP2A Mut3 was targeted towards theElk1 binding site, and CIP2A Mut4 targeted the binding site fortranscription factor Pax5, while CIP2A Mut5 and CIP2A Mut6 targeted thebinding sites for Ets1 and Elk1.

FIG. 11 depicts a luciferase assay in human cervical carcinoma (HeLa)transfected with Mut1, Mut2, Mut3, Mut4, Mut5 and Mut6 or the wild-typepromoter (CIP2A−171/+70) and assayed for luciferase activity after 48hours. Transfection efficiency was normalized by co-transfection withpRL-TK (Renilla expression vector). The means±S.D. are from threedifferent experiments, each experiment performed in triplicate(***p>0.001, **p>0.01, *p>0.05 with CIP2A−171/+70 compared to thecontrol pGL4 basic vector and Mutant compared to CIP2A−171/+70 wild-typeconstruct).

FIG. 12 depicts a luciferase assay in human endometrial carcinoma (ECC1)transfected with Mut1, Mut2, Mut3, Mut4, Mut5 and Mut6 or the wild-typepromoter (CIP2A−171/+70) and assayed for luciferase activity after 48hours. Transfection efficiency was normalized by co-transfection withpRL-TK (Renilla expression vector). The means±S.D. are from threedifferent experiments, each experiment performed in triplicate(***p>0.001, **p>0.01, *p>0.05 with CIP2A−171/+70 compared to thecontrol pGL4 basic vector and Mutant compared to CIP2A−171/+70 wild-typeconstruct).

FIG. 13 depicts the in vitro binding of Ets1 and Elk1 to the proximalpromoter region of the CIP2A gene. Electrophoretic mobility shift assay(EMSA) was performed with nuclear extracts (9 μg) ECC-1 cells that wasincubated with the wild-type (WT) probe (−138 to −107) from human CIP2Agene as described in Materials and Methods. In competition experiments,a 100-fold molar excess of the designated probes were utilized todemonstrate the specificity of each binding reaction. Arrows indicatethe formation of specific protein-DNA complexes. The experiment wasrepeated twice with similar results.

FIG. 14 depicts Ets1 and Elk1 binding to the proximal promoter of theCIP2A gene. Nuclear extracts (9 μg) from ECC-1 were incubated with WTprobe (−138 to −107) from the CIP2A gene along with 5 μg of antibodydirected towards Ets1, Elk1, or Ets1 and Elk1 together. The negativecontrol consisted of rabbit-pre-immune control IgG. DNA-protein complexformations are designated by solid arrows.

FIG. 15A depicts the effect of Ets1, Elk1 siRNA on CIP2A mRNAexpression. HeLa cells were transiently transfected with 100 nM of Ets1siRNA (SEQ ID NOs: 30, 31) or Elk1 (SEQ ID NOs: 32, 33) or Ets1/Elk1(SEQ ID NOs: 30, 31, 32, 33) or siRNA-scramble as the negative control.The siRNA transfected cells showed a decrease in CIP2A mRNA expressionlevels.

FIG. 15B depicts the Western blot analysis of the HeLa cells transectedwith siRNA against the Ets1 and Elk1 mRNA. siRNA treatment reduces CIP2Aprotein, without altering the GAPDH level, indicating specificity.

FIG. 16A depicts the effects of Ets1 siRNA on Ets1 mRNA expression. HeLacells were transiently transfected with 100 nM of Ets1 siRNA (SEQ IDNOs: 30, 31) or Ets1/Elk1 (SEQ ID NOs: 30, 31, 32, 33) or siRNA-scrambleas the negative control. The siRNA transfected cells showed a decreasein Ets1 mRNA expression levels.

FIG. 16B depicts the effect of Elk1 siRNA on Elk1 mRNA expression. HeLacells were transiently transfected with 100 nM of Elk1 siRNA (SEQ IDNOs: 32, 33) or Ets1/Elk1 (SEQ ID NOs: 30, 31, 32, 33) or siRNA-scrambleas the negative control. The siRNA transfected cells showed a decreasein Elk1 mRNA expression levels.

FIG. 17 depicts the effects of Ets1 siRNA on CIP2A protein expression inPC-3 (prostate cancer cells) in a Western blot analysis. GAPDH is usedas a loading control. PC-3 cells were transiently transfected with 100nM of Ets1 siRNAs (SEQ ID NO: 30 and SEQ ID NO: 31) or siRNA-scramble asthe negative control (Csi; Dhramacon; catalog number: D-001206-13-20).The siRNA transfected cells showed a decrease, in Ets1 proteinexpression as well as CIP2A protein expression.

FIG. 18 depicts the ectopic expression of ETS1 and ELK1 together rescuesCIP2A expression against 3′-UTR siRNA treatment. HeLa cells weretransiently transfected with 100 nM of ETS1 and ELK1 3′-UTR siRNAtogether or non-targeting (NT) siRNA as the negative control. ETS1 andELK1 cDNA were cloned into pCDN4-His Max Topo expression vector(Invitrogen, K864-20) utilizing the primers mentioned in Table 7,generating Ets1-Topo and Elk1-Topo. Sequences of the clones wereverified and 1 μg was utilized for ectopic expression in HeLa cellsfollowing 3′-UTR siRNA treatment. Cells transfected with the emptyvector served as a negative control. (A) Western blot analysis of CIP2A,Ets1, Elk1 and GAPDH protein expression levels were analyzed 72 hoursafter transfection and (B) qRT-PCR was conducted at 48 hours aftertransfection to confirm rescue of CIP2A mRNA **p<0.01.

FIG. 19 depicts the ChIP analysis of Ets1 and Elk1 binding to the CIP2Apromoter. (A) Schematic representation of the Ets1 and Elk1 bindingsites within the CIP2A proximal promoter region. The primers utilizedfor amplification of DNA region are noted in Table 6. (B) ChIP analysisof the Ets1 and Elk1 association with the CIP2A promoter. Mouse IgGserves as a negative control. Region 1 is specific for a regioncontaining the Ets1 and Elk1 binding sites in the CIP2A promoter, whileregion 2 is a distal part of the CIP2A gene which is devoid of the Ets1and Elk1 binding sites.

FIG. 20 depicts the ChIP analysis of Ets1 and Elk1 binding to the CIP2Apromoter. (A) Schematic representation of the Ets1 and Elk1 bindingsites within the proximal promoter region. The primers utilized foramplification of DNA region are noted in Table 4. ChIP qPCR analysis ofEts1 and Elk1 association with the CIP2A promoter for region 1 in (B)HeLa and (C) ECC-1 and region 2 for (D) HeLa and (E) ECC-1 are shown.Mouse IgG serves as a negative control. Region 1 is specific for theregion containing the Ets1 and Elk1 binding sites in the CIP2A promoter,while region 2 is a distal part of the CIP2A gene which is devoid ofEts1 and Elk1 binding sites. The fold enrichment from IgG, Ets1 and Elk1immunoprecipitation are shown and were calculated relative to input as %input. The fold change in occupancy was calculated by setting the foldenrichment of IgG to 1. The results are from two different experiments,each experiment performed in duplicate (***p<0.001, **p<0.01, *p<0.05with Ets1, Elk1 compared to the control IgG).

FIG. 21 depicts the CIP2A, Ets1 and Elk1 protein expression levels inhuman cervical tumor samples. Six (6) different matched pair of humancervical tumor samples (T) with adjacent normal tissue (N) were obtainedand used in this series of experiments. Protein extraction was performedusing protocol as described in Experimental Methods and Protocols.CIP2A, Ets1 and Elk1 protein expression were monitored using Westernblot analysis. Patient pathological characteristics of patient cervicalcarcinoma tissue samples were described in Table 1. The proteinexpression levels of (A) CIP2A, (B) Elk1, (C) Ets1 and Actin (i.e.,β-actin; used as a loading control) were monitored by western blotting.

DETAILED DESCRIPTION OF THE INVENTION

The present invention can be better understood from the followingdescription of preferred embodiments, taken in conjunction with theaccompanying drawings. It should be apparent to those skilled in the artthat the described embodiments of the present invention provided hereinare merely exemplary and illustrative and not limiting.

DEFINITIONS

Various terms used throughout this specification shall have thedefinitions set out herein.

As used herein, the term “A,” “T,” “C”, “G” and “U” refer to adenine,thymine, cytosine, guanine, uracil as a nucleotide base, respectively.

As used herein, the term “siRNA” refers to a small interfering RNA. RNAinterference refers to the process of sequence-specificpost-transcriptional gene silencing in a cell or an animal mediated byshort interfering RNA.

As used herein, the term “siRNA targeted against Ets1” or “siRNAtargeted against Elk1” refers to siRNA specifically promote degradationof Ets1 mRNA or Elk1 mRNA via sequence-specific complementary basepairings.

As used herein, the term “target sequence” refers to a contiguousportion of the nucleotide sequence of an mRNA molecule of a particulargene (i.e., CIP2A gene). The target sequence of a siRNA of the presentinvention refers to a mRNA sequence of that gene that is targeted by thesiRNA by virtue of its complementarity of the anti-sense strand of thesiRNA to such sequence and to which the anti-sense strand hybridizeswhen brought into contact with the mRNA. The mRNA sequence may includethe number of nucleotides in the anti-sense strand as well as the numberof nucleotides in a single-stranded overhang of the sense strand, ifany.

As used herein, the term “complementary” refers to the ability of afirst polynucleotide to hybridize with a second polynucleotide.

As used herein, the term “double-stranded RNA” (“dsRNA”) refers to acomplex of ribonucleic acid molecules having a duplex structurecomprising two anti-parallel and substantially complementary nucleicacid strands.

As used herein, the term “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa.

As used herein, the term “palindromic” refers to a nucleotide sequencewhich is the same when read either in the 5′-3′ direction or in the3′-5′ direction.

As used herein, the term “ectopic expression” refers to expression of agene having a modified promoter when such a gene is being introducedinto an organism for gene expression.

As used herein, the term “blunt” refers that there are no unpairednucleotides at that end of the dsRNA (i.e., no nucleotide overhang). A“blunt ended” dsRNA is a dsRNA that has no nucleotide overhang at eitherend of the molecule.

As used herein, the term “anti-sense strand” refers to the strand of adsRNA which includes a region that is substantially complementary to atarget sequence.

As used herein, the term “sequence complementarity” refers to a sequenceregion on the anti-sense strand that is substantially complementary to asequence. Where the sequence complementarity is not fully complementaryto the target sequence, the mismatches are most tolerated in theterminal regions and, if present, are generally in a terminal region orregions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′terminus. Most preferably, the mismatches are located within 6, 5, 4, 3,or 2 nucleotides of the 5′ terminus of the anti-sense strand and/or the3′ terminus of the sense strand.

As used herein, the term “sense strand,” refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the anti-sense strand.

As used herein, the term “introducing into a cell”, when referring to adsRNA, means facilitating uptake or absorption into the cell, as isunderstood by those skilled in the art. Absorption or uptake of dsRNAcan occur through unaided diffusive or active cellular processes, or byauxiliary agents or devices. The meaning of this term is not limited tocells in vitro; a dsRNA may also be “introduced into a cell”, whereinthe cell is part of a living organism. In such instance, introductioninto the cell will include the delivery to the organism. For example,for in vivo delivery, dsRNA can be injected into a tissue site oradministered systemically. In vitro introduction into a cell includesmethods known in the art such as electroporation and lipofection.

As used herein, the terms “silencing” and “inhibiting the expressionof”, in as far as they refer to the CIP2A gene refers to at leastpartial suppression of the expression of the CIP2A gene, as manifestedby a reduction of the amount of mRNA transcribed from the CIP2A gene.Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to CIP2A genetranscription, e.g. the amount of protein encoded by the CIP2A genewhich is secreted by a cell or found in solution after lysis of suchcells.

As used herein, the term “CIP2A gene” refers to Cancerous InhibitorPhosphate 2A gene, the nucleotide sequence of which is listed underGenbank accession numbers NM_020890.2, the disclosure of which isincorporated herein by reference.

As used herein, the term “deletion constructs” in the context of CIP2Apromoter refers to the varying size fragments of the CIP2A promoter. Thedeletion constructions of CIP2A are obtained by sequentially deletion ofthe full-length CIP2A promoter by PCR and they are harbored in a pGL4basic vector. The PCR is performing by maintaining the binding site ofthe reverser primer while altering the binding sites of a forwardprimer, thus creating CIP2A promoter fragments of varying sizes.

As used herein, the term “promoter” refers to a region of the DNA thatfacilitates the transcription of a particular gene.

As used herein, the term “˜3 kb promoter fragment” refers to the−2379/+70 fragment of the CIP2A promoter and the nucleotide sequence ofwhich is provided in FIG. 4.

As used herein, the term “Ets1” (E-twenty-six-specific) refers to amember of the family of transcription factors involved in regulating theexpression of genes involved in apoptosis, development, differentiation,proliferation and transformation. The Ets family binds to the GGAA/Tcore motif and nucleotides flanking the core binding site increasespecificity for Ets family member binding. The term “variant” refers tothe different forms of the same protein (i.e., isoform) which differs inmolecular weight and structure. For example, Ets1 variant 1 has amolecular weight of 55 kDa and represents the long isoform. Thenucleotide sequence of the Ets1 mRNA variant 1 is listed under GenBankaccession numbers NM_001143820.1, the disclosure of which isincorporated herein by reference. Ets1 variant 2 has a molecular weightof 51 kDa, and represents the short isoform. The nucleotide sequence ofthe Ets1 mRNA variant 2 is listed under GenBank accession numbersNM_005238.3, the disclosure of which is incorporated herein byreference.

As used herein, the term “Elk1” (Ets like kinase 1) refers to one of theEts family member transcription factors known to regulate the expressionof c-fos in cooperation with serum response elements (SRE). Elk1 showspreference for binding the consensus sequence CCGGAAGTR often with asecond transcription factor SRE. Elk1 variant 1 and Elk1 variant 2encode the same protein. However, Elk1 variant 2 differs from Elk1variant 1 in that it lacks an internal 5′-UTR. The nucleotide sequenceof the Elk1 mRNA variant 1 is listed under GenBank accession numbersNM_001114123.1, the disclosure of which is incorporated herein byreference. The nucleotide sequence of the Elk1 mRNA variant 2 is listedunder GenBank accession numbers NM_005229.3, the disclosure of which isincorporated herein by reference.

As used herein, the term “pGL4” refers to the vector that encodes theluciferase reporter gene luc2 (Photinus pyralis) with high expression.The pGL4 vector is optimized for recombinant expression in mammaliancells. pGL4 basic has multiple cloning sites that allow the cloning ofthe promoter of interest.

As used herein, the term “luciferase activity” refers to the use ofluciferase reporter to assess the transcriptional activity of aparticular gene construct in a cell under the control of a promoter ofinterest.

As used herein, the term “cervical cancer” refers to the cancer thatstarts at the cervix (lower part of the uterus) and spreads to the topof the vagina.

As used herein, the term “endometrial cancer” refers to the cancer thatstarts at the endometrium, the tissue lining of the uterus. Endometrialcancer is also referred to as uterine cancer.

As used herein, the terms “attenuate”, “treat” and “treatment” refer torelief from or alleviation of pathological processes mediated by CIP2Aexpression. In other words, relief from or alleviate at least onesymptom associated with such condition, or to slow or reverse theprogression of such condition.

The term “attenuate gene expression” refers to the use of siRNA moleculeto down regulate the expression of target gene mRNA such as Ets1 orElk1, which thereby leads to reduced expression of these genes.

As used herein, the term “quantitative PCR” refers to the quantitativepolymerase chain reaction. Quantitative PCR is a means for quantifyingthe amount of template DNA present in the original mixture, usuallyachieved by the addition of a known amount of a target sequence that isamplified by the same primer set but can be differentiated, usually bysize, at the end of the reaction.

As used herein, the term “real-time PCR” refers to the real-timepolymerase chain reaction. Real-time PCR is a method for the detectionand quantitation of an amplified PCR product based on a fluorescentreporter dye; the fluorescent signal increases in direct proportion tothe amount of PCR product produced and is monitored at each cycle, ‘inreal time’, such that the time point at which the first significantincrease in the amount of PCR product correlates with the initial amountof target template.

As used herein, the term “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” refers toa carrier for administration of a therapeutic agent.

As used herein, the term “therapeutically effective amount” refers to anamount that provides a therapeutic benefit in the treatment, prevention,or management of pathological processes mediated by CIP2A expression oran overt symptom of pathological processes mediated by CIP2A expression.

In one aspect, the present invention provides characterization of thehuman CIP2A promoter including both the proximal and minimal proximalpromoter. The present inventors show that the GGAA-binding transcriptionfactors (e.g., Ets1 and Elk1) function together to regulate CIP2A geneexpression in human cervical, endometrial and liver carcinoma cells. The5′ flanking minimal proximal promoter of the CIP2A gene consists ofputative binding sites for Ets1 and Elk1 in forward and reverseorientations.

In another aspect, the present invention provides cloning of varioushuman CIP2A promoters. In doing so, the present inventors identified two(2) Ets1/Elk1 binding sites in reverse orientations in the CIP2Apromoter required for CIP2A gene expression. Without wishing to be boundby a theory, the presence of two (2) Ets1 binding sites is speculated toenhance the association between Ets/Ets homodimers by stabilizing theinteraction and thus overcoming the auto-inhibitory domain of Etstranscription factors. The presence of two (2) Ets1 binding sites mayprovide an explanation as to the surprising finding that both Ets1 andElk1 are required for regulating CIP2A gene expression in cells.

In another aspect, the present invention provides that Ets1 and Elk1binding are essential for CIP2A basal expression in urogenital cancercells, cervical cancer cells, endometrial cancer cells and livercarcinoma cells. siRNA knock-down of Ets1 and Elk1 together decreaseCIP2A gene transcription, whereas knock-down of Ets1 or Elk1 alone doesnot decrease CIP2A gene transcription. Ectopic expression of Ets1 andElk1 together increase CIP2A expression. There is a direct correlationbetween the levels of CIP2A and the levels of Ets1 and Elk1. The presentfindings indicate that the binding of Ets1 and Elk1 to the proximalCIP2A promoter is required for CIP2A expression in cancer cells.

In another aspect, the present invention provides the minimal promoterconstruct that provides gene regulation for CIP2A. In one embodiment,the present invention identifies and characterizes the transcriptionalfactors that control CIP2A expression. Utilizing functional deletionconstructs of CIP2A gene, the present inventors discovered that theregion between −123 and −95 of the CIP2A gene promoter is essential forthe basal transcription in human cervical (HeLa), liver (HepG2), andendometrial carcinoma (ECC-1) cells. Transcriptional factors Ets1 andElk1 are required for regulating the transcription of the CIP2A gene inhuman cervical and endometrial carcinoma cells.

The present invention therefore provides a therapeutic strategy of usingsiRNA targeted against Ets1 and Elk1 as a treatment for endometrial andcervical cancers. The present inventors provided the first elucidationfor the transcriptional regulation of CIP2A in female urogenitalcancers. The disclosed data find support in human CIP2A gene basalproximal and minimal proximal promoter in ECC-1 and HeLa cell lines. Itis proposed that the GGAA binding transcription factors such as Ets1 andElk1 work in a co-operative manner in the basal regulation of CIP2Aexpression in human cervical and endometrial carcinoma cells.

In one embodiment, the data support the minimal proximal promoter ofCIP2A lies between −123/+70. Both Ets1 and Elk1 bind to this palindromicregion to regulate the CIP2A expression. Without being bound by atheory, it is believed that the presence of palindromic Ets1 bindingsites enhances an association between Ets1/Ets1 heterodimers by creatinga stable interaction in the DNA binding domain, which overcomes theauto-inhibitory domain the Ets DNA binding region. The Ets-DNA bindingis flanked by N- and C-terminal inhibitory regions that causeauto-inhibition and impair DNA binding.

In one embodiment, the present invention provides an observation thatCIP2A luciferase activity was decreased when the Mut4 construct (Pax5mutation) was expressed in ECC-1 cells when compared to the proximalpromoter (−171/+70). However, expression was equal to the activity ofthe minimal proximal promoter region (−123/+70), which suggests thatPax5 may not be a major transcription factor regulating CIP2A in ECC-1cells. This difference may be explained by a cooperative interactionbetween Pax5 and Elk1. It is speculated that the recruitment of Elk1 andNet by Pax5 may form a functional ternary complex in the B-cell specificpromoter. Association with Pax5 may alter the Ets1 binding motif fromGGAA to GGAG upon their interaction. It is surprising to note that it ispossible that different regions of the CIP2A gene regulate basaltranscription in different cell types.

In another embodiment, the present invention provides the observationthat Ets1 binding is essential. Mutations in the Ets1 binding siteswithin the CIP2A gene promoter result in a decrease in the CIP2Apromoter activity (e.g., in human gastric adenocarcinoma (AG 1478)cells).

In another embodiment, the present invention further provides anobservation that there is a requirement for both Ets1 and Elk1 in CIP2Atranscription in HeLa and ECC-1 cells (i.e., urogenital cancers). Thepresent discovery is in sharp contrast to that of Khanna et al. (PLOS2011, 6: 1-13) who suggested that Ets1 transcription factor aloneregulates CIP2A expression levels in human gastric (AG1478) and prostate(PC-3 and LNCaP) carcinoma cells. Contrary to Khanna et al., the presentdiscovery illustrates Ets1 and Elk1 are both required in cervical andendometrial cells, evidencing that there is cell-type specificregulation of CIP2A.

In one aspect, the present invention provides an isolated doublestranded short interfering ribonucleic acid (siRNA) molecule thatsilences expression of Ets1 mRNA. In another aspect, the presentinvention provides an isolated double stranded short interferingribonucleic acid (siRNA) molecule that silences expression of Elk1 mRNA.

The mechanism of action of siRNA is understood by one skilled in theart. Interfering RNA (RNAi) generally refers to a single-stranded RNA ordouble-stranded RNA (dsRNA). The dsRNA is capable of targeting specificmessenger RNA (mRNA) and silencing (inhibiting) the expression of atarget gene. During the process, dsRNA is enzymatically processed intoshort-interfering RNA (siRNA) duplexes of 21 nucleotides in length. Theanti-sense strand of the siRNA duplex is then incorporated into acytoplasmic complex of proteins (RNA-induced silencing complex or RISC).The RISC complex containing the anti-sense siRNA strand also binds mRNAwhich has a sequence complementary to the anti-sense strand—allowingcomplementary base-pairing between the anti-sense siRNA strand and thesense mRNA molecule. The mRNA molecule is then specifically cleaved byan enzyme (RNase) associated with RISC resulting in specific genesilencing. For gene silencing or knock down (i.e., mRNA cleavage) tooccur, anti-sense RNA (i.e., siRNA) has to become incorporated into theRISC. This represents an efficient process that occurs in nucleatedcells during regulation of gene expression. When an anti-sense DNAmolecule is introduced into a cell, it targets specific mRNA throughbase-pairing of the anti-sense DNA molecule to its RNA target.

For purposes of this application, the anti-sense strand of the siRNA maycomprise a contiguous nucleotide sequence, where the base sequence ofthe anti-sense strand has sequence complementarity to the base sequenceof contiguous nucleotide sequence of corresponding length contained inthe mRNA sequence of the targeted mRNA (e.g., Ets1 or Elk1 mRNA).Complementary includes complete base-pairing match or a few base-pairingmis-match.

In one embodiment, the anti-sense strand of the siRNA molecule comprisesor consists of a sequence that is 100% complementary to the targetsequence or a portion thereof. In another embodiment, the anti-sensestrand of the siRNA molecule comprises or consists of a sequence that isat least about 90%, 95%, or 99% complementary to the target sequence ora portion thereof. For purposes of this application, the anti-sensestrand of the siRNA molecule comprises or consists of a sequence thatspecifically hybridizes to the target sequence or a portion thereof soas to inhibit the target mRNA expression.

Without wishing to be bound by a theory, siRNA-mediated RNA interferencemay involve two-steps: (i) an initiation step, and (ii) an effectorstep. In the first step, input siRNA is processed into small fragments,such as 21-23-nucleotide ‘guide sequences’. The guide RNAs can beincorporated into a protein-RNA complex which is capable of degradingmRNA, the nuclease complex, which has been called the RNA-inducedsilencing complex (RISC). The RISC complex acts in the second effectorstep to destroy mRNAs that are recognized by the guide RNAs throughbase-pairing interactions. siRNA involves the introduction by any meansof double stranded RNA into the cell which triggers events that causethe degradation of a target RNA. siRNA is a form of post-transcriptionalgene silencing. One of skilled in the art would understand thepreparation and utilization of siRNA molecules. (See, e.g., Hammond etal., Nature Rev Gen 2: 110-119 (2001); Sharp, Genes Dev 15: 485-490(2001), the disclosure of which are incorporated herein by reference intheir entireties).

Methods for preparing and isolating siRNA are known in the art (See,e.g., Smabrook et al., Molecular Cloning, A Laboratory Manual (2^(nd)Ed., 1989), the disclosure of this is herein incorporated by referencein its entirety). In one embodiment, siRNA are chemically synthesized,using any of a variety of techniques known in the art, such as thosedescribed in Wincott et al., Nucl. Acids Res., 23:2677-2684 (1995); andWincott et al., Methods Mol. Bio., 74:59 (1997). The synthesis of thesiRNA makes use of common nucleic acid protecting and coupling groups,such as dimethoxytrityl at the 5′-end and phosphoramidites at the3′-end. Suitable reagents for siRNA synthesis, methods for RNAdeprotection, and methods for RNA purification are known to those ofskill in the art. Small scale syntheses or large scale syntheses can beconducted using suitable synthesizer and protocols that are recognizedin the industry. Preferably, siRNA molecules are chemically synthesized.

siRNA molecules can also be synthesized via a tandem synthesistechnique, wherein both strands are synthesized as a single continuousstrand separated by a cleavable linker that is subsequently cleaved toprovide separate strands that hybridize to form the siRNA duplex. Thetandem synthesis of siRNA can be readily adapted to both multi-well ormulti-plate synthesis platforms as well as large scale synthesisplatforms employing batch reactors, synthesis columns, and the like.Alternatively, siRNA molecules can be assembled from two distinctoligonucleotides, wherein one oligonucleotide comprises the sense strandand the other comprises the anti-sense strand of the siRNA. For example,each strand can be synthesized separately and joined together byhybridization or ligation following synthesis and/or deprotection. Incertain other instances, siRNA molecules can be synthesized as a singlecontinuous oligonucleotide fragment, where the self-complementary senseand anti-sense regions hybridize to form a siRNA duplex having hairpinsecondary structure.

In one embodiment, siRNA comprises a double stranded region of about 15to about 30 nucleotides in length. Preferably, siRNA has about 20-25nucleotides in length. The siRNA molecules of the present invention arecapable of silencing the expression of a target sequence in vitro and invivo.

In one embodiment, the siRNA comprises a hairpin loop structure. Inanother embodiment, the siRNA has an overhang on its 3′ or 5′ endsrelative to the target RNA which is to be cleaved. The overhang may be2-10 nucleotides long. In one embodiment, the siRNA does not have anoverhang (i.e., blunted).

In another embodiment, the siRNA molecule may contain one modifiednucleotide. In yet another embodiment, the siRNA may comprise one, two,three four or more modified nucleotides in the double-stranded region.Exemplary modified siRNA molecule includes, but not limited to, modifiednucleotides such as 2′-O-methyl (2′OMe) nucleotides, 2′-deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy nucleotides, 2′-O-(2-methoxyethyl) (MOE)nucleotides, and the like. The preparation of modified siRNA is known byone skilled in the art.

Because CIP2A overexpression has been proposed to correlate with thedrug resistance in tumor treatment. Specifically, CIP2A is overexpressedin breast cancer cells and is correlated with the development ofdoxorubicin resistance (Choi et al., FEBS Lett. 2011; 585: 755-60).Another exemplary observation comes from the finding that overexpressionof CIP2A in hepatocellular carcinoma cells (HCC) PLC5 leads toresistance to bortezomib (Chen et al., Oncogene 2010; 29: 6257-66). Thepresent observation that there is a correlation in the CIP2A proteinexpression, Ets1 and Elk1 in six (6) matched pair of human cervicaltumor samples further substantiates the important role of Ets1 and Elk1.The present invention provides a therapeutic approach of employingsiRNAs to block the Ets1 and Elk1 expressions and thus reduces the CIP2Aexpression and attenuate the tumor development.

In one aspect, the present invention provides two exemplary anti-sensestrand siRNAs that hybridize to the Ets1 mRNA so as to increasedegradation of Ets1 mRNA (and consequently Ets1 protein expression). Inone embodiment, the present invention provides a first exemplaryanti-sense strand siRNA (SEQ ID NO: 30) that hybridzises to Ets1 mRNA.This first exemplary anti-sense strand siRNA hybridizes to the Ets1 mRNAvariant 1 (SEQ ID NO: 34) or Ets1 mRNA variant 2 (SEQ ID NO: 35).

In another embodiment, the present invention provides a second exemplaryanti-sense strand siRNA (SEQ ID NO: 31) that hybridzises to Ets1 mRNA.This second exemplary anti-sense strand siRNA hybridizes to the Ets1mRNA variant 1 (SEQ ID NO: 36) or Ets1 mRNA variant 2 (SEQ′ID NO: 37).

In another aspect, the present invention also provides two exemplary ananti-sense strand siRNAs that hybridize to the Elk1 mRNA so as toincrease degradation of Elk1 mRNA (and consequently Elk1 proteinexpression). In one embodiment, the present invention provides a firstexemplary anti-sense strand siRNA (SEQ ID NO: 32) that hybridizes toElk1 mRNA. This first exemplary anti-sense strand siRNA hybridizes tothe Elk1 mRNA variant 1 (SEQ ID NO: 38) or Ets1 mRNA variant 2 (SEQ IDNO: 39).

In another embodiment, the present invention provides a second exemplaryanti-sense strand siRNA (SEQ ID NO: 33) that hybridizes to Elk1 mRNA.This second exemplary anti-sense strand siRNA hybridizes to the Elk1mRNA variant 1 (SEQ ID NO: 40) or Elk1 mRNA variant 2 (SEQ ID NO: 41).

In one embodiment, the present siRNA molecule targeting Ets1 is capableof silencing the Ets1 mRNA at least about 40%-100% of the expression ofthe target sequence relative to the corresponding unmodified (i.e.,control) siRNA sequence. In another embodiment, the present siRNAmolecule targeting Elk1 is capable of silencing the Elk1 mRNA at leastabout 40%-100% of the expression of the target sequence relative to thecorresponding unmodified (i.e., control) siRNA sequence.

The present siRNA molecule targeting Ets1 and Elk1 can be used todown-regulate or inhibit the expression of CIP2A. The CIP2A expressionis inhibited by at least about 40%-100%.

Our present finding is further supported by the ChIP study. The ChIPresults support the hypothesis that both Elk1 and Est-1 are associatedwith the CIP2A gene promoter in cervical cells (e.g., HeLa cells). BothElk1 and Ets1 transcriptional factors are found to be associated withCIP2A gene promoter, albeit the Elk1 binding is stronger than that ofEts1 in associating with the CIP2A gene promoter. The present finding isentirely unexpected. To the best of the present inventors' knowledge,this represents the first reported observation that demonstrates thatElk1 can regulate CIP2A expression as well as the co-operative manner ofboth Elk1 and Ets1 in regulating CIP2A gene.

siRNA may conveniently be delivered to a target cell through a number ofdirect delivery systems. For example, siRNA may be delivered viaelectroporation, lipofection, calcium phosphate precipitation, plasmids,viral vectors, viral nucleic acids, phage nucleic acids, phages,cosmids, or via transfer of genetic material in cells or carriers suchas cationic liposomes. In one embodiment, transfection of siRNA mayemploy viral vectors, chemical transfectants, or physico-mechanicalmethods such as electroporation and direct diffusion of DNA. The siRNAdelivery methods are known in the art and readily adaptable for use.(See, e.g., Wolff, J. A., et al., Science, 247, 1465-1468, (1990); andWolff, J. A. Nature, 352, 815-818, (1991)).

In one aspect, the present invention provides a pharmaceuticalcomposition containing siRNAs targeted against Ets1 and Elk1 for thetreatment of cervical cancer and endometrial cancer. The pharmaceuticalcomposition comprises the siRNAs as therapeutic agents for inhibitingCIP2A gene activity and a pharmaceutical acceptable carrier.Pharmaceutically acceptable carriers include, but are not limited to,excipients such as inert diluents, disintegrating agents, bindingagents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to aid absorption in thegastrointestinal tract.

Pharmaceutical compositions containing siRNA may be administered to amammal in vivo to treat cancer. In one embodiment, the pharmaceuticalformulation includes a dosage suitable for oral administration. Inanother embodiment, the pharmaceutical formulation is designed to suitvarious means for siRNA administration. Exemplary means include uptakeof naked siRNA, liposome fusion, intramuscular injection via a gene gun,endocytosis and the like. These administration means are well known inthe art.

The present invention provides a means for attenuating (i.e.,inhibiting) the CIP2A gene using siRNA targeting against Ets1 and Elk1.It is known that increased CIP2A may be associated with the developmentof drug resistance in various cancer cells. For example, doxorubicinresistance in breast cancer cells and bortezomib resistance inhepatocellular carcinoma cells. The present invention provides a methodof reducing drug resistance in cancer cells by reducing Ets1 or Elk1expression levels. Specifically, the present invention provides a methodof using siRNA targeted against Ets1 and Elk1 in attenuating the CIP2Aexpression in urogenital cancers, including cervical cancer andendometrial cancer. The combined use of siRNA to attenuate Ets1 and Elk1represents a prognostic tool in treating human cervical carcinoma.

In one embodiment, the siRNA is administered to a human with atherapeutic effective amount of siRNA targeting Ets1 or Elk1transcriptional factors. The specific amount that is therapeuticallyeffective can be readily determined by monitoring the CIP2A mRNA levels.Inhibition of Ets1 or Elk1 mRNAs is conveniently achieved by usingqRT-PCR, Northern blot analysis and other techniques known to those ofskill in the art such as dot blots, in situ hybridization, and the like.The inhibition level is comparing the target gene expression to thecontrol. A detectable inhibition can be about 40%-100%. Preferably, the% inhibition may be 80%, 90% or 100%. The therapeutic effective amountmay be determined by ordinary medical practitioner, and may varydepending on factors known in the art, such as the patient's history andage, the stage of pathological processes mediated by CIP2A expression.

The following examples are provided to further illustrate variouspreferred embodiments and techniques of the invention. It should beunderstood, however, that these examples do not limit the scope of theinvention described in the claims. Many variations and modifications areintended to be encompassed within the spirit and scope of the invention.

EXPERIMENTAL STUDIES Example 1 Cloning of CIP2A Promoters

CIP2A cDNA has been cloned by Hoo et al. (2002), and was identified asan auto-antigen over-expressed along with p62 in hepatocellularcarcinoma patients sera. Thereafter, several authors recently reportedincreased expressions of CIP2A in various types of cancers cells andtumor samples from patients ranging from gastric, breast, oral,prostate, cervical, esophageal squamous cell carcinoma, non-small celllung carcinoma, early-stage tongue cancer, acute myeloid leukemia,chronic myeloid leukemia and invasive rheumatoid arthritis (Li et al.,2008; Côme et al., 2009; Khanna et al., 2009; Katz et al., 2010; Vaaralaet al., 2010; Liu et al., 2011; Qu et al., 2010; Dong et al., 2010;Lucas et al., 2011; Wang et al., 2011; Lee et al., 2011). However, thetranscriptional elements regulating the expression of CIP2A inurogenital cancers have remained uncharacterized.

The CIP2A nucleotide gene sequence has been deposited in GenBank(GenBank accession no. AC092693.8), the disclosure of which isincorporated by reference. In this study, we specifically examined thegenomic structure of the CIP2A promoter and the role of itstranscriptional regulation. To do so, we prepared a CIP2A promoterconstruct upstream from that of the CIP2A transcription start site (i.e.ATG), and prepared 5′-deletion clones as well as the full-length 2.4kilo-basepairs (kbp) basal promoter clone. The first exon was foundbetween +1 and +70 nucleotides (FIG. 1). The 5′ flanking region upstreamof the transcription start site (TSS)+1 region is predicted to harborthe promoter region for transcriptional regulation of CIP2A.

We used PCR approach with a specific primer pair (SEQ ID NOs: 10 and 11)(See, Table 3) designed to amplify nucleotide positions 7301-4854 of theCIP2A gene with the GenBank Accession Number AC 092693.8 bearing the BACclone RP11-161J9. The resulting PCR CIP2A cloned fragment has anucleotide sequence listed in SEQ ID NO: 1 (See, Table 3 and FIG. 4).

The cloned DNA consisted of a promoter fragment containing ˜3 kb that isupstream of the CIP2A transcription start site (i.e., ATG). Theamplified ˜3 kb promoter fragment was purified using gel extractionprotocol. We then used restriction enzymes to insert the amplified ˜3 kbpromoter fragment into the luciferase reporter construct (i.e., pGL4.10[luc2] luciferase reporter which contains only the luciferase codingregion and no regulatory element) (See, FIG. 2). The resultingluciferase reporter construct containing the ˜3 kb CIP2A promoterfragment is named CIP2A−2379/+70 plasmid (See, FIG. 3).

Example 2 Nucleotide Sequence of the ˜3 kb CIP2A Promoter Fragments

The ˜3 kb promoter fragment was sequence-verified. We used the SEQ IDNOs: 10 and 11 (Table 3) against the forward and reverse strands and thesequencing was performed on a CEQ 8000 Genomic analyzer (BeckmanCoultier). We compared the sequence of the ˜3 kb promoter fragment (SEQID NO: 1) with the nucleotide sequence of the CIP2A gene (GenBankAccession Number, AC 092693.8) and verified that there was no mutationintroduced through PCR cloning. The nucleotide sequence (SEQ ID NO: 1)of our ˜3 kb promoter fragment is listed in FIG. 4.

Example 3 Additional CIP2A Promoter Constructs (˜1.4 kb, ˜1 kb, ˜0.5 kb,˜0.2 kb and ˜0.1 kb)

So far, we have successfully cloned the ˜3 kb CIP2A promoter in theluciferase reporter vector. In this study, we sought to identify theminimal proximal promoter region from CIP2A transcription start site(i.e., ATG) required for constitutive expression of CIP2A. We alsoidentified the respective role of the putative transcriptional sitespresent on this CIP2A promoter region. To do so, we took the initiativeto generate eight (8) additional CIP2A promoter constructs (in additionto the ˜3 kb CIP2A promoter construct).

We cloned a total of nine (9) CIP2A promoter luciferase reporterconstructs with various CIP2A promoter lengths from ˜2.4 kb to ˜0.1 kb.As described in Example 2, we used forward primer (SEQ ID NO: 10) andreverse primer (SEQ ID NO: 11) to clone out CIP2A−2379/+70 CIP2A clone.We utilized the CIP2A−2379/+70 construct (SEQ ID NO: 1) as the templatefor generating the various CIP2A promoter fragments. All the CIP2Apromoter fragments were generated. The CIP2A clones includedCIP2A−1452/+70 construct (SEQ ID NO: 2), CIP2A−941/+70 construct (SEQ IDNO: 3), CIP2A−428/+70 construct (SEQ ID NO: 4), CIP2A−284/+70 construct(SEQ ID NO: 5), CIP2A−213/+70 construct (SEQ ID NO: 6), CIP2A−171/+70construct (SEQ ID NO: 7), CIP2A−123/+70 construct (SEQ ID NO: 8), andCIP2A−95/+70 construct (SEQ ID NO: 9).

Table 3 summarizes the forward primers and reverse primers used ingenerating various CIP2A clones. The forward primer and reverse primersused in preparing CIP2A−1452/+70 (SEQ ID NO: 2) were SEQ ID NOs: 12 and13 respectively. The forward primer and reverse primers used inpreparing CIP2A−941/+70 (SEQ ID NO: 3) were SEQ ID NOs: 14 and 15respectively. The forward primer and reverse primers used in preparingCIP2A−428/+70 (SEQ ID NO: 4) were SEQ ID NOs: 16 and 17 respectively.The forward primer and reverse primers used in preparing CIP2A−284/+70(SEQ ID NO: 5) were SEQ ID NOs: 18 and 19 respectively. The forwardprimer and reverse primers used in preparing CIP2A−213/+70 (SEQ ID NO:6) were SEQ ID NOs: 20 and 21 respectively. The forward primer andreverse primers used in preparing CIP2A−171/+70 (SEQ ID NO: 7) were SEQID NOs: 22 and 23 respectively. The forward primer and reverse primersused in preparing CIP2A−123/+70 (SEQ ID NO: 8) were SEQ ID NOs: 24 and25 respectively. The forward primer and reverse primers used inpreparing CIP2A−95/+70 (SEQ ID NO: 9) were SEQ ID NOs: 26 and 27respectively. All of the nine (9) CIP2A clones and their primer pairsare listed in Table 3. The primers utilized for PCR amplificationconsisted of NheI and XhoI restriction enzymes in the forward andreverser primers for cloning into the luciferase reporter vector. Thesequences of all the nine (9) CIP2A constructs were verified bysequencing.

FIG. 5 summarizes the prepared luciferase reporter constructs harboringvarious CIP2A promoter fragments. Table 3 summarizes the preparedadditional CIP2A promoter luciferase constructs that include 1.4 kb, ˜1kb, ˜0.5 kb, ˜0.2 kb and ˜0.1 kb CIP2A promoter (upstream fromtranscription start site). The nucleotide sequence of these CIP2Apromoter constructs is included in FIG. 4. The clone names of thevarious CIP2A constructs are also indicated.

Example 4 Identification of CIP2A Proximal and Minimal Proximal PromoterRegions

In order to identify functional transcription factor binding sites inthe 5′ flanking region of the CIP2A gene promoter, we prepared a seriesof PCR deletion clones for CIP2A promoter using the pGL4 basicluciferase vector. FIG. 5 shows the constructed luciferase reporterconstructs carried various CIP2A promoter fragments, with the pGL4luciferase vector. In order to identify the CIP2A promoter regionresponsible for constitutive expression of CIP2A, we transientlytransfected all the 5′ CIP2A deletion constructs into either humancervical carcinoma cells (HeLa cells) or human liver hepatobalstomacells (HepG2 cells). Moreover the cells were also transientlytransfected with pRL-TK vector (FIG. 6) carrying the Renilla luciferase(RL) under with thymidine kinase (TK) promoter and enhancer elements fornormalization of transfection efficiency. The fold change in relativeluciferase activity (RLA) of individual deletion clone was compared withthat of pGL4 basic vector (i.e., pGL4.10 [luc2] luciferase reporterwhich contains only the luciferase coding region and no regulatoryelement), which serves as the negative control.

(i) Human Cervical Carcinoma and Liver Hepatobalastoma

We found that the full-length CIP2A promoter (−2379/+70) possessed a50-fold increase in HeLa cells (FIG. 7) and a 5-fold increase in HepG2cells (FIG. 8) when compared to the basic vector. The CIP2A−95/+70 cloneshowed no activity above background in these cells (FIGS. 7 and 8). Theactivity of promoter construct −171/+70 was similar to the full-lengthconstruct −2379/+70 of the CIP2A gene promoter. Based on the deletionconstructs (FIG. 5), the construct containing the region −171/+70displayed a 57- and 5-fold increase in RLA, while the promoter region−123/+70 showed 11- and 2-fold increase in RLA in HeLa and HepG2 cellswhen compared to pGL4 basic vector. These data indicate that clone−123/+70 contains the minimal proximal promoter activity of the humanCIP2A gene.

We observed that the DNA fragment −941/+70 showed the highest (253- and20-fold) increase in relative luciferase activity (RLA) in these cells(FIGS. 7 and 8). These data suggest there may be enhancer and/orcorepressor binding sites upstream of the minimal proximal promoter,which further regulate the basic CIP2A activity mediated by the −123/+70region.

In sum, these results indicate that the clone −123/+70 contain theminimal proximal promoter of the human CIP2A gene. Clones CIP2A−213/+70,CIP2A−284/+70, CIP2A−428/+70 and CIP2A−1452/+70 showed similarluciferase activity as that of CIP2A−171/+70 in HeLa and HepG2 cells(FIGS. 7 and 8).

(ii) Human Endometrial Carcinoma Cells

In a separate series of study, we assessed the CIP2A gene transcriptionin human endometrial (ECC-1) carcinoma cells. Deletion constructsharboring the promoter regions −123/+70, −171/+70, −428/+70, −941/+70and −2379/+70 (FIG. 5) were utilized.

A 71- and 283-fold increase in RLA was observed with CIP2A−123/+70,CIP2A−171/+70 clones in comparison with CIP2A−95/+70 clone whichdisplayed only a 5-fold increase in RLA when compared to pGL4 Basic(FIG. 9). As was observed in HeLa and HepG2 cells, the CIP2A−941/+70construct showed the highest activity, while the luciferase activity offull-length construct −2379/+70 was similar to CIP2A−171/+70.

Altogether, these results identify the region between −123 to −95 as theminimal proximal promoter region. Such region is believed to beessential for regulating the transcription of human CIP2A gene promoterin human cervical, liver and endometrial carcinoma cells. The minimalproximal region corresponds with the nucleotide 5046-5018 of the BACclone RP11-161J9 with CIP2A gene and GenBank Accession No. AC 092693.8.While the region between −123 and −95 constitutes the minimal proximalpromoter essential for CIP2A expression in human cervical, liver andendometrial carcinoma cells, the region between −171 and −95 containsthe proximal promoter. The data are summarized in Table 2.

Example 5 The ˜200 bp CIP2A Proximal Promoter Contains Multiple PutativeTranscription Factor(s) Binding Sites

The CIP2A promoter is speculated to regulate through binding oftranscription factors to the transcriptional sites present on theproximal promoter region. We next sought to identify putativetranscription factor(s) binding sites present within the ˜200 bp CIP2Aproximal promoter. To do this, we performed a bioinformatics analysis ofthe ˜200 bp human CIP2A promoter construct in order to identify putativetranscription factor (s) binding sites in this fragment. Specifically,we analyzed the proximal promoter region (−171 to −95) of the humanCIP2A promoter for potential transcription factor binding sites.

We utilized two (2) bioinformatics programs: (i) ALIBABA 2.0(www.gene-regulation.com) and (ii) ALGEN-PROMO(www.alggen.lsi.upc.es/cgi-bin/promo_v3/promo). With these programs, wepredicted eight (8) potential transcription factor(s) binding sites.Binding sites for GRα (glucocorticoid receptor alpha), RARα (retinoicacid alpha), Pax5, Ets1, Elk1, AP-2 and Sp1 were identified within the−171 to +1 region (See, FIG. 1). FIG. 1 depicts the overlapping sharedtranscription factor(s) binding sites present on the ˜200 bp human CIP2Apromoter. The binding sites for the Ets-1 and Elk1 transcription factorswere identified in reverse orientations in region between −110/−118 and−127/−137 (FIG. 1).

Example 6 Deciphering the Specific Transcription Factor Important forRegulating the Transcription of CIP2A Promoter: Site-DirectedMutagenesis Studies

Based on our results from the 5′ deletion analysis and computationalscreening, we identified potential binding sites for NF-κB, RARα, Ets1,Elk1 and Pax5 transcription factor(s) in the region between −171 and −95of the CIP2A gene promoter (the basal proximal promoter region). We nextutilized PCR based site-directed mutagenesis, point mutations (baseshighlighted in bold and underlined) or deletions (bases highlighted inbold with double strike) were introduced within the transcription factorbinding sites (FIG. 10). The CIP2A−171/+70 construct was used in all themutagenesis studies (FIG. 10).

We have constructed several human CIP2A promoter constructs havingspecific mutant promoter sites. In a first mutant CIP2A promoterconstruct, the transcription factor binding site for NF-κB inCIP2A−171/+70 construct was mutated (point mutations) to generate themutant CIP2A Mut1 (SEQ ID NO: 58). In a second mutant CIP2A promoterconstruct, the first binding site for Ets1 was altered to generate themutant CIP2A Mut2 (SEQ ID NO: 59). In a third mutant CIP2A promoterconstruct, the first binding site for Elk1 was deleted to generate themutant CIP2A Mut3 (SEQ ID NO: 60). In a fourth mutant CIP2A promoterconstruct, the binding site for Pax-5 was altered with base substitutionto yield the mutant CIP2A Mut4 (SEQ ID NO: 61). In a fifth mutant CIP2Apromoter construct, the second Ets1 binding site in CIP2A promoterregion between −110 to −118 was deleted to generate the mutant CIP2AMut5 (SEQ ID NO: 62). In a sixth mutant CIP2A promoter construct, thesecond Elk1 binding site in CIP2A promoter region was substituted toyield the mutant CIP2A Mut6 (SEQ ID NO: 63). (See, FIG. 10).

Transfection of the CIP2A Mut2 and CIP2A Mut3 construct displayed a 3-4fold reduced activity in the HeLa cells and a 7-16 fold reduction inactivity in the ECC-1 cells compared to the wild-type CIP2A−171/+70construct (FIGS. 11 and 12). In contrast, the mutation in the NF-κBbinding site (CIP2A Mut1 construct) did not affect the basal luciferaseactivity in the HeLa and ECC-1 cells compared to the wild type constructCIP2A−171/+70 (FIGS. 11 and 12).

Mutation in the Pax5 binding site (CIP2A Mut4) did not affect thetranscription of the CIP2A gene promoter in the HeLa cells (FIG. 11),but decreased the CIP2A transcription by 2.5-fold in ECC-1 cells (FIG.12), indicating cell-type specificity.

Moreover, when the HeLa and ECC-1 cells were transfected with CIP2AMut5, a 9-16 fold loss in CIP2A promoter activity was observed.Similarly, the CIP2A Mut6 displayed a 13-39-fold decrease in luciferaseactivity compared to the wild-type construct CIP2A−171/+70 in HeLa andECC-1 cells (FIGS. 11 and 12).

Altogether, these results suggest that the transcription factor bindingsites for Ets1 and Elk1 are crucial for the basal, constitutivetranscription of CIP2A gene in human cervical and endometrial cancercells.

Example 7 In Vitro Binding of Ets1 and Elk1 Transcriptional Proteins toCIP2A Minimal Proximal Promoter Region

Our data showed a requirement for Ets1 and Elk1 proteins in drivingCIP2A gene transcription. In this study, we performed an ElectrophoreticMobility Shift Assay (EMSA) to examine the binding of Ets1 and Elk1transcriptional factors to the CIP2A promoter region. EMSA, alsocommonly known as mobility shift electrophoresis or gel shift assay,represents an affinity electrophoresis technique that is used to studyprotein-DNA interactions. We used EMSA to determine if the Ets1 and Elk1proteins bind to CIP2A minimal promoter constructs.

In this study, we first synthesized a wild-type (WT) probe harboring theEts1 and Elk1 binding sites in the forward and reverse orientation from−138 to −107 bp of the CIP2A gene promoter with a consensusoligonucleotide for Ets1 and Elk1.

From ECC-1 cells, we next prepared nuclear extracts. As shown in FIG.13, addition of the WT probe in the presence of nuclear extracts fromECC-1 cells displayed four (4) protein-DNA complexes (FIG. 13, lane 2).Competition with a 100-fold molar excess of unlabeled WT probe resultedin a complete inhibition of all four (4) protein-DNA complexes (FIG. 13,lane 3), indicating specificity. Competition with a 100-fold molarexcess of unlabeled mutant probe, in which the palindromic binding sitesfor Ets1 and Elk1 were mutated, did not abolish the fourth protein-DNAcomplex, though the first three complexes were inhibited (FIG. 13, lane4).

From these competition results, we speculated that it was the fourthDNA-protein complex that served as the binding site for Ets1 and Elk1.To confirm the loss of the fourth DNA-protein complex as the bindingsite for Ets1 and Elk1, a 100-fold excess molar competition wasperformed with the Ets1 and Elk1 consensus sequences. Addition of a100-fold excess molar of Ets1 and Elk1 consensus sequences led to theloss of the fourth protein-DNA complex (FIG. 13, lanes 5-6).

Moreover, the pattern of protein-DNA complexes observed with the labeledEts1 and Elk1 consensus probe were similar to those observed with the WTprobe of the CIP2A gene (FIG. 13, compare lane 2 with lanes 8 and 9).These results indicate that the transcription factors Ets1 and Elk1 bindto the −138 to −107 region of the CIP2A gene and regulate itstranscription in endometrial carcinoma cells.

In a separate series of experiments, we performed a gel-super shiftanalysis to further confirm our results. As a negative control,pre-immune IgG was utilized (FIG. 14, lane 3) and no shift was detected.Addition of Ets1 antibody to the nuclear extract from ECC-1 cells in thepresence of the WT probe caused a shift in the protein DNA complex (FIG.14, lane 4). Interestingly, in the presence of Elk1 antibody, theintensity of the protein-DNA complex was greatly enhanced rather than ashift (FIG. 14, lane 5). In the presence of both Ets1 and Elk1antibodies, we detected a shift and an increase in the intensity of theprotein-DNA complex (FIG. 14, lane 6). These gel-shift assay resultsfurther confirm that Ets1 and Elk1 bind to the palindromic sequence(−138 to −107) of the CIP2A promoter.

All together, these studies show the in vitro binding of Ets1 and Elk1transcription proteins to the CIP2A proximal promoter region.

Example 8 siRNA-Targeted Reductions of Ets1 and Elk1 Leads to a Decreasein CIP2A mRNA Expression

Our results from site-directed mutagenesis identified the Ets1/Elk1palindromic binding sites within the ˜200 bp CIP2A promoter essentialfor regulating CIP2A transcription in human cervical and endometrialcarcinoma cells and demonstrated the in vitro association of Ets1/Elk1binding to CIP2A gene promoter. In order to analyze directly the role ofindividual transcription factor(s) Ets1 or Elk1 in regulating thetranscription of CIP2A gene, human cervical carcinoma cells weretransfected with siRNA specific towards Ets1 (SEQ ID NO: 30, 31), Elk1(SEQ ID NO: 32, 33) or Ets1/Elk1 together (Table 5). A significant 40%decrease in CIP2A mRNA expression levels was observed when HeLa cellswere transfected with siRNA towards Ets1/Elk1 together, in contrastthere was no significant effect in altering CIP2A mRNA expression levelswhen siRNA specific towards Ets1 or Elk1 was utilized (FIG. 15A).Moreover the specificity of siRNA utilized for knock-down of target genewas significant as there was a 3-fold decrease in Ets1 and Elk1 mRNAexpression levels (FIG. 16) on treatment with siRNA specific for Ets1,Elk1 or Ets1/Elk1 together.

Additionally, in order to corroborate the results from knock-downstudies the effect of siRNA on endogenous expression of CIP2A proteinwas analyzed in human cervical carcinoma cells (HeLa) cells 72 hoursafter transfection. As shown in FIG. 15B decreased CIP2A protein levelswere observed with both Ets1/Elk1 together, while there was no change inCIP2A protein levels in the presence of either Ets1 or Elk1individually. These results clearly demonstrate the direct role ofEts1/Elk1 together in regulating the basal transcription of CIP2A genein human cervical carcinoma cells (HeLa).

In order to confirm if the CIP2A regulation by siRNA targeting Ets1 andElk1 is cell type specific, we repeated the experiments using adifferent cell type (e.g., prostate cancer cells). We transfected acocktail of siRNAs targeting against Ets1 into a human prostatecarcinoma cell type (PC-3). The cocktail of siRNAs targeting Ets1 iscomposed of two (2) siRNA having nucleotide sequences set forth in SEQID NO: 30 and SEQ ID NO: 31.

As shown in FIG. 17, we observed a significant decrease in CIP2A proteinexpression in the PC-3 cells following siRNA treatment. These dataindicate Ets1 alone is sufficient in regulating CIP2A in prostate cancercells. The CIP2A regulation in prostate cancer cells and gastric cancercells has been previously reported by Khanna et al. (2011) to requireEts1. Contrary to prostate cancer cells and gastric cancer cells, Ets1alone is not sufficient in regulating CIP2A in cervical cancer cells orendometrial cells, but require both Ets1 and Elk1 (See, FIG. 15).Altogether, these data suggest CIP2A transcriptional regulation is celltype specific—while Ets1 alone regulates CIP2A in prostate and gastriccancer cells, both Ets1 and Elk1 are required in cervical andendometrial cancer cells.

Add-Back Studies—Specificity of Ets1 and Elk1 in Expression of CIP2A

To corroborate our observations, we completed an add-back assay toconfirm the specificity for Ets1 and Elk1 in basal expression of CIP2A.In this series of studies, we transfected HeLa cells with eithernon-targeting siRNA (FIG. 18A, lane 1) or siRNA against the 3′-UTRregions of ETS1 and ELK1, effectively depleting the cells of endogenousEts1 and Elk1 protein (FIG. 18A, lane 2).

The nucleotide sequences for the 3′ UTR ETS1 are GGUUGGACUCUGAAUUUUG(SEQ ID NO: 64) that binds to the nucleotides 1599-1617 on theNM_001143820.1 (the disclosure of which is incorporated herein byreference). The nucleotide sequences for the 3′ UTR ETS1CCCCAAGGUUAAAUACAA (SEQ ID NO: 65) that binds to the nucleotides3166-3184 on the NM_001143820.1 (the disclosure of which is incorporatedherein by reference). The nucleotide sequences for the 3′ UTR ELK1 areGCGGUUUAUUUAUUUAUUU (SEQ ID NO: 66) that binds to the nucleotide1868-1886 on NM_991114123.1 (the disclosure of which is incorporatedherein by reference). The nucleotide sequences for the 3′ UTR ELK1 areCUGCCAUUUUGAUAGUAUA (SEQ ID NO: 67) that binds to the nucleotides2420-2437 on NM_001114123.1 (the disclosure of which is incorporatedherein by reference).

We co-transfected cells with either empty vector (FIG. 18A, lanes 1 and2) or vectors over-expressing ETS1 gene or ELK1 gene or ETS1 and ELK1genes together (FIG. 18A, lanes 3, 4, 5), which were resistant to ETS1and ELK1 3′-UTR siRNA and analyzed cell lysates by western analysis. Asignificant decrease in CIP2A protein levels was observed in HeLa cellsupon treatment with both ETS1 and ELK1 3′-UTR siRNA (FIG. 18A, lane 2)when compared to non-targeting siRNA (FIG. 18A, lane 1).

Furthermore ectopic expression of either ETS1 gene or ELK1 gene alonedid not rescue CIP2A expression in HeLa cells (FIG. 18A, lanes 3, 4)treated with ETS 1 and ELK1 3′-UTR siRNA. The loss of CIP2A protein wasrescued in HeLa cells when ETS1 gene and ELK1 gene were over-expressedtogether (FIG. 18A, lane 5) in the presence of ETS1 and ELK1 3′-UTRsiRNA. CIP2A mRNA was significantly decreased when cells were treatedwith ETS1 and ELK1 3-UTR siRNA (FIG. 18B, comparing column 1 and column2), when analyzed by qRT-PCR, and CIP2A expression was not recoveredwhen ETS1 gene or ELK1 gene were over-expressed individually (FIG. 18B,comparing column 3 and column 4 to column 1). CIP2A expression wasreturned to normal levels when both ETS1 gene and ELK1 gene wereover-expressed (FIG. 18B, comparing column 5 to column 1). These resultsconfirm the specificity of Ets1 and Elk1 transcription factors inregulating the basal transcription of CIP2A.

Example 9 In Vivo Association of Ets1 and Elk1 with the CIP2A GenePromoter

To assess the in vivo association of Ets1 and Elk1 to the CIP2A genepromoter, a chromatin immunoprecipitation (ChIP) analysis was performed.The cross-linked protein-DNA was immunoprecipitated with antibodiesagainst Ets1, Elk1. Mouse pre-immune IgG was used as a control. In aseries of study, the amplification of a 123 bp fragment harboring theEts1 and Elk1 binding sites within the CIP2A promoter was detected inthe immunoprecipitates obtained in HeLa cells when the Ets1 or Elk1antibodies were used (FIG. 19B, region 1), while there was noamplification in the IgG control.

A second region was amplified that does not contain Ets1 or Elk1 bindingsites in the CIP2A promoter, 2379 bp downstream of the ATG start site.The 278 bp fragment was visible in the input sample obtained from theHeLa cell line (FIG. 19B, region 2) while amplification was not seen inDNA obtained with antibody precipitation. These results demonstrate thatthe transcription factor(s) Ets1 and Elk1 associate with CIP2A genepromoter in HeLa cells.

In a separate series of study, we continued to confirm our in vitro dataand have used chromatin immunoprecipitation (ChIP) analyses andquantitative PCR to assess the direct in vivo association of Ets1 andElk1 with the CIP2A gene promoter. Cross-linked protein-DNA wasimmunoprecipitated with antibodies against Ets1, Elk1 or pre-immune IgG.

We used two primer sets to examine the specificity of Ets1 and Elk1binding (FIG. 20A). Region 1 contains the two binding sites for Ets1 andElk1, while region 2 has no binding sites for the transcription factors.We found that Ets1 immunoprecipitation (IP) resulted in a significant3-fold increase in binding to the CIP2A promoter at region 1, while IPwith the Elk1 antibody resulted in a significant 2-fold increase inbinding to the CIP2A promoter compared to IgG in HeLa cells (FIG. 20B).Similarly Ets1 and Elk1 IP from ECC-1 cells showed a significant 2-foldincrease in binding to CIP2A promoter occupancy compared to control IgG(FIG. 20C). In contrast, the Ets1 and Elk1 immunoprecipitates did notamplify CIP2A promoter fragment in the region between −2379 to −2101 ineither cell lines. These results demonstrate that Ets1 and Elk1associate with CIP2A gene promoter in vivo in cervical and endometrialcarcinoma cells.

Example 10 Expression Levels of CIP2A, Ets1, and Elk1 in Human CervicalPatient Samples

To correlate our findings in human cervical patient samples, six (6)matched pairs of cervical tumor and normal adjacent tissue (NAT) werepurchased. The protein levels of CIP2A, Ets1, Elk1 and Actin wereidentified via Western blot analysis. Increased expression of CIP2Aprotein levels were observed in all six tumor samples compared to theNAT samples (FIG. 21A). Similarly, four (4) of the tumor samples showedan increase in Elk1 (FIG. 21B) and Ets1 (FIG. 21C) protein levels whencompared to NAT samples. In all tumor samples, Ets1, Elk1, or both wereover-expressed. Together, these results substantiate the observationthat Ets1 and Elk1 regulate the basal transcription of CIP2A in humancervical tumors.

Experimental Methods and Protocols

A) Cell Culture

The human cervical carcinoma cell line HeLa was grown in DMEMsupplemented with 10% fetal calf serum (Invitrogen, Carlsbad, Calif.)and penicillin-streptomycin (Sigma, St. Louis, Mo.) at a finalconcentration of 100 μg/ml. The hepatocellular carcinoma cells HepG2were maintained in DMEM with 10% FCS and 10 μg/ml gentamicin(Invitrogen, Carlsbad, Calif.). The endometrial carcinoma cells ECC-1were grown in RPMI-1640 supplemented with 5% FCS (Invitrogen, Carlsbad,Calif.). All cells were grown at 37° C. in a 5% CO₂ incubator.

B) Human Cervical Carcinoma Tissue Samples

Six matched pair of human cervical carcinoma tissue samples werepurchased from ILS Bio, LLC (Chestertown, Md.). These tissue sampleswere analyzed for their protein expression levels of CIP2A, Ets1, andElk1. The description of the tissue sample is provided in Table 1.

C) Construction of CIP2A Luciferase Reporter Vector and 5′ DeletionAnalysis

The BAC clone RP11-161J9 (Rosewell Parker Cancer Institute Human BAClibrary) harboring the 5′ flanking region of CIP2A gene (GenBankaccession no AC092693.8) was utilized to design the primers forconstruction of CIP2A promoter-luciferase plasmids. DNA isolated fromHeLa cells were utilized as templates to generate PCR fragments usingTaq polymerase (Takara), which were further cloned into the reportervector at the NheI-XhoI poly cloning sites by incorporating thecorresponding restriction sties in the forward and reverse primers. Thefull length construct −2379/+70 luciferase promoter consists ofapproximately 2.4 Kbp region upstream and 70 bp downstream oftranscription start site (TSS) was cloned into the pGL4.10 [luc2] vector(Promega, Madison, Wis.). Similarly PCR amplified promoter regions−1452/+70, −941/+70, −428/+70, −284/+70, −213/+70, −171/+70, −123/+70and −95/+70 were cloned in NheI-XhoI sites of pGL4-basic vector. Thenucleotide sequence of the clones was verified by sequencing.

D) Transient Transfection and Luciferase Assay

Cells were seeded in 6-well plates at a density of 5×10⁵ cells/well forHeLa, HepG2 and 8×10⁵ for Ecc1 cells, 24 hours before transfection.Transfection was performed utilizing Lipofectamine 2000 reagent(Invitrogen) following manufactures recommendations. In each experiment,2 μg of control vector (pGL4-basic without CIP2A promoter insert, emptyvector) or the reporter vector (CIP2A full length promoter fragment orsequentially deleted CIP2A PCR fragments in pGL4-basic vector) wasco-transfected along with 250 ng of pRL-TK (Reniella luciferase,Promega) as an internal control. Following incubation with DNA complexfor 4 h cells were feed with 2 mL of fresh growth medium for anadditional 44 hours. Luciferase assay was utilizing a 384 well roboticplate reader (EnVision, Perkin Elmer).

E) Identification of Potential Putative Transcription Factor BindingSites in CIP2A Gene Promoter

Potential transcription factor(s) binding sites within the CIP2A genepromoter was screened with the assistance of computer programs such asALGEN-PROMO or ALIBABA 2.0 programs (www.gene-regulation.com).

F) Site-Directed Mutagenesis

The −171/+70 CIP2A promoter fragment was used to generate mutant clonesof CIP2A promoter. The Quickchange lightning site-directed mutagenesiskit (Stratagene) was utilized to generate mutants CIP2A Mut1-CIP2A Mut6.Primers for introduction of point mutations or deletions were designedas instructed by the manufacturer. The nucleotide sequence of themutated clones was verified by sequencing. The promoter activity of themutated clones was assayed by transient transfection and luciferaseassay as detailed in previous section.

G) Electrophoretic Mobility Shift Assay (EMSA) and Gel Super-Shift

Nuclear extracts were prepared from ECC-1 cells. 1×10⁷ cells were seededin 75 cm² flasks 24 h before nuclear proteins were extracted utilizingthe nuclear complex CO-IP kit (Active Motif, 54001) as instructed by themanufacturer. The wild-type and the mutant probes were synthesized asdouble stranded oligonucleotides (Integrated DNA technology) from the−138 to −107 region of the CIP2A gene promoter. Consensusoligonucleotides for Ets1 and Elk1 were synthesized based on thesequence data from Santa Cruz Biotechnology (Santa Cruz, Calif.) andPanomics (Affymetrix, CA). The sequences of the probes utilized were:WT-5′-GACTTCCGGAGCCCGACCGGATCCGGAAGCTT-3′ (SEQ ID NO: 68);Mutant-5′-GAAAATTTAAGCCCGACCGGATAAATTTACTT-3′ (mutated bases shown inbold text) (SEQ ID NO: 69); Et1s-5′-GATCTCGAGCAGGAAGTTCGA-3′ (SEQ ID NO:70); Elk1-5′-TTTGCAAAATGCAGGAATTGTTTTCACAGT-3′ (SEQ ID NO: 71). All theprobes were labeled with biotin using the Biotin 3′-end DNA labeling kit(Thermo Scientific, 89818) according to the manual. Nine micrograms ofthe nuclear extract was utilized for the binding reactions. The EMSAbinding reactions were performed at room temperature for 30 min andconsisted of the nuclear extract in 1× binding buffer (50% glycerol, 100mM MgCl₂, 1 μg/μl Poly (dI-dC), 1% NP-40, 1 M KCl, 200 mM EDTA and 5 μMDNA probe). The mixture was run on 8% non-denaturing polyacrylamide gelsin 0.5×Tris Borate-EDTA buffer at 170 V. The protein-DNA complexes werethen transferred to Hybond-N+ nylon membrane using the Trans-Blotsemi-dry method (Bio-Rad, CA) and cross-linked using the SpectrolinkerXL-1000 UV crosslinker (Spectronics Corporation, NY). Detection ofbiotin-labeled DNA was performed using the LightShift chemiluminsecentEMSA kit (Thermo Scientific, 20148) and visualized by exposure to acharge-couple device camera (GE ImageQuant LAS 4000).

For competition EMSA, 100-fold molar excess of the cold, mutant orconsensus oligonucleotide was added to the EMSA binding reaction. Forthe gel-Supershift assay, following the incubation of the nuclearextracts with the 32 bp WT CIP2A promoter fragment for 30 min, 5 μg ofEts1 antibody (Abcam, ab124282), 5 μg of Elk1 antibody (Epitomics,1277-1) and/or the two antibodies (anti-Ets1 and Elk1) were added to thebinding reaction and the mixture incubated at RT for an additional 30min. The pre-immune IgG (Millipore, 12-371) was utilized as negativecontrol in the Supershift assay. The mixture was fractionated on 5%non-denaturing polyacrylamide gel. Transfer and detection was performedas described above.

H) Ets1/Elk1 siRNA Knockdown, Overexpression and CIP2A ExpressionAnalysis

In order to assess the direct effect of Ets1/Elk1 in regulating thetranscription of CIP2A gene promoter, human siRNA specific towards(i.e., targeted against) Ets1 (Dharmacon, Lafayette, Colo.; J-003887-06,J-003887-08), Elk1 (Dhramacon, Lafayette, Colo.; J-003885-06,J-003885-08) or Ets1/Elk1 together were utilized. siGENOME non-targetingsiRNA pool 1 (Dharmacon, D-001206-13) was used as negative control.Human cervical carcinoma cell line HeLa were seeded at a density of5×10⁵ cells/well were transiently transfected with 100 nM of each of thetargeted siRNA or in combination utilizing Lipofectamine 2000(Invitrogen). RNA and protein were isolated 24 hours and 72 hours aftertransfection for CIP2A expression levels.

One microgram (1 μg) of the RNA was used in the reverse transcriptionreaction along with 4 U of Omniscript reverse transcriptase (Qiagen,Valencia, Calif.), 1 μM oligo-dT primer (Qiagen), 0.5 mM dNTP (Qiagen),10 U of RNase inhibitor (Qiagen) and 1×RT buffer (Qiagen). Reversetranscription was performed at 37° C. for 1 hour with a final incubationat 93° C. for 5 min for inactivation of reverse transcriptase. Twomicroliters (2 μl) of the RT-product was used in the real time PCRreaction. The Quantitect SYBR green PCR kit (Qiagen) was utilized andPCR was performed according to the manufacturer's instructions using theStratagene MXPRO 3000 real time RT cycler (Agilent Technologies, SantaClara, Calif.). Each PCR reaction consisted of 50% (v/v) of 2×SYBR greenmaster mixes and 0.2 μM gene-specific forward and reverse primers.Quantification of glyceraldehyde-6-phosphate dehydrogenase (GADPH) wasused to normalize the relative expression levels of CIP2A, Ets1 and Elk1mRNA. Each experiment was performed in duplicates and repeated at leasttwice. The primer sequences used for qPCR are as follows:

(i) CIP2A: (SEQ ID NO: 48) Forward: TGCCTGCTTGAAGTCCTTG (SEQ ID NO: 49)Reverse: TAGTCGTGTGAGTTTCTGTCC (ii) GAPDH: (SEQ ID NO: 50)Forward: TGGGCTACACTGAGCACCAG (SEQ ID NO: 51)Reverse: GGGTGTCGCTGTTGAAGTCA (iii) Ets1: Qigen product no: PPH01781B(iv) Elk1: Qiagen product no: PPH00140B

For the siRNA rescue experiment, 5×10⁵HeLa cells seeded in 6 well plateswere transiently transfected with ETS1 3′-UTR siRNA and ELK1 3′-UTRsiRNA (Dharmacon). siGENOME non-targeting siRNA pool 1 (Dharmacon) wasutilized as the negative control. Transfections were performed with 100nM of the targeted 3′-UTR siRNA in combination or the non-targeting poolwith Lipofectamine RNAiMax (Invitrogen) as described by themanufacturer. Forty-eight hours after 3′-UTR siRNA treatment, cells weretransiently transfected with 1 μg of Ets1-Topo and Elk1-Topo mammalianexpression vector encoding respective cDNAs utilizing LipofectamineRNAiMax (Invitrogen). HeLa cells transfected with empty vector served asa negative control. Protein was extracted 72 h after transfection forCIP2A analysis. Rabbit anti-CIP2A (Novus, NB100-68264), rabbit anti-Ets1(Abcam, ab124282), rabbit-anti-Elk1 (Epitomics, 1277-1) and rabbitant-GAPDH (Abcam, ab9385) were utilized for western blotting.

(I) Chromatin Immunoprecipitation Assay (ChIP Assay)

Chromatin immunoprecipitation was performed using 1-2×10⁷ HeLa or ECC-1cells, which were treated with 37% formaldehyde (Sigma, F1635) at 1%final concentration, vol/vol for 10 min at room temperature tocross-link proteins to DNA. After cross-linking the cells were washedtwice with 1× ice-cold PBS containing protease inhibitor cocktail(Sigma, P8340). The cells were collected and centrifuged at 700×g for 2min, re-suspended in 1 ml of SDS-lysis buffer with protease inhibitorcocktail. The cells were then sonicated twice with a bioruptor(Diagenode, UCD200) at high power, with 30 s on/off pulse for 15 mins(break up DNA to ˜500 bp fragments). The cell lysate was centrifuged at10,000×g for 15 min at 4° C. and the supernatant was further subjectedto enzymatic digestion utilizing micrococcal nuclease (New EnglandBiolabs, M0247) for 15 mins at 37° C. The enzyme activity wasinactivated by adding 0.5 M ETDA and incubated on ice for 10 mins. 25 μlof the DNA fraction was kept aside as input for PCR. The remaining DNAfraction was precleared using a mixture of 35 μl of protein G andprotein A agarose beads each (50% slurry, Millipore, 16-201, 16-157) for2 hours at 4° C.

Immunoprecipitation was performed by adding antibodies towards Ets1(Abcam, ab124282), Elk1 (Epitomics, 1277-1) or mouse IgG (Millipore,12-371) as the negative control. The immunocomplex was precipitated byincubation with 70 μl of protein A/G agarose beads for 2 h at 4° C. Theprotein/DNA complex was eluted using 200 μl of elution buffer (1% SDS,0.1 M NaHCO₃) from the beads. Cross-linking of protein-DNA was reversedby adding 10 μl of 5 M NaCl at 65° C. for 2-3 hours. The DNA waspurified using spin columns (Promega, A9281) and 5 μl of the DNA wasused in the qPCR reaction for amplification of 198 bp or 298 bp of theCIP2A promoter region (FIG. 5A). qPCR reactions were performed utilizingthe primers in Table 6.

(J) Western Blot Analysis

Protein was isolated from cells, by initially washing the cells in 1×ice-cold PBS and re-suspending the cells in 250 μl of RIPA buffersupplemented with protease inhibitor cocktail (Sigma). Fifty microgram(50 μg) of protein was fractioned on 10% or 12% SDS-PAGE and transferredto nitrocellulose membrane, blocked at 4° C. overnight in 5% non-fatmilk in TBS and incubated with rabbit anti-CIP2A (Novus), rabbitanti-Elk1 (Cell Signaling, Danvers, Mass.), rabbit anti-Ets1 (Millipore,Billerica, Calif.) and rabbit anti-GAPDH (Abcam, Cambridge, Mass.) at 4°C. overnight. Blots were washed with 1×TBST and incubated withanti-rabbit secondary HRP at room temperature for 1 hour. Detection ofsignal was performed by adding chemiluminsecent substrate (Pierce,Rockford, Ill.) and visualized by exposure to a charge-couple devicecamera (GE lmageQuant LAS 4000, Piscataway, N.J.).

(K) Statistical Analysis

Statistical analysis was performed for calculating the significantdifferences in luciferase activity between constructs, effect of siRNAin knock-down of CIP2A mRNA expression and effect of Ets1, Elk1overexpression in CIP2A mRNA by one way randomized analysis of variance(ANOVA) and Newam-Keuls test with significance level of p<0.05.

All publications and patents cited in this specification are hereinincorporated by reference in their entirety. Various modifications andvariations of the described composition, method, and systems of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments andcertain working examples, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.

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TABLE 1 Pathological Characteristics of Human Cervical Carcinoma TissueSamples Identification No. Cervical Cancer Phenotype Donor Race 19203 NGeorgian 19203 D Undifferentiated Carcinoma Grade III Georgian 19306 NGeorgian 19306 D Adenosquamous Cell Carcinoma Grade II Georgian 19205 NGeorgian 19205 D Squamous Cell Carcinoma (SSC) Grade II Georgian 19246 NGeorgian 19246 D Squamous Cell Carcinoma (SSC) Grade II Georgian 24726 NVietnamese 24726 D Adenocarcinoma Grade III Vietnamese 28016 NVietnamese 28016 D Adenocarcinoma Grade III Vietnamese N is normalsubject; D is disease subject.

TABLE 2 Identification of Human CIP2A Basal Proximal Promoter Foldincrease in Relative 5′ Deletion luciferase activity (RLA) ConstructHeLa HepG2 ECC-1 pGL4 Basic 1 1 1 CIP2A −95/+70 0.4 0.4 5.3 CIP2A−123/+70 11.0 1.8 71.0 CIP2A −171/+70 57.0 5.0 208.0 CIP2A −213/+70 39.04.0 — CIP2A −284/+70 54.0 6.0 — CIP2A −428/+70 58.0 12.0 210.0 CIP2A−941/+70 253.0 30.0 426.0 CIP2A −1452/+70 63.0 6.0 — CIP2A −2379/+7065.0 4.0 134.0 Human cervical carcinoma cells (HeLa), liverhepatobalstoma cells (HepG2), and endometrial carcinoma cells (ECC-1)were transfected with various CIP2A promoter constructs. Luciferaseactivity was assayed 48 hours after transfection. Fold increase inrelative luciferase activity (RLA) was compared with pGL4 basic (valueset as 1). Normalization in transfection efficiency was performed byco-transfection with pRL-TK (Renilla expression vector). Mean ± S.D. arefrom three different experiments, each performed in triplicate.

TABLE 3 Primer Sequences Utilized for CIP2A 5′ Deletion Clones SEQ ID NOCIP2A clone Forward Primer Reverser Primer 1 CIP2A −2379/+705′-GCTAGCaaactggaaattaaaagcgtgagc-3′ 5′-CTCGAGcctctgacttcacggctttgt-3′(SEQ ID NO: 1) (SEQ ID NO: 10) (SEQ ID NO: 11) 2 CIP2A −1452/+705′-GCTAGCctcccttggccagattttacctaat-3′ 5′-CTCGAGcctctgacttcacggctttgt-3′(SEQ ID NO: 2) (SEQ ID NO:12) (SEQ ID NO: 13) 3 CIP2A −941/+705′-GCTAGCtacaatttctacatcctggtttttaaagc-3′5′-CTCGAGcctctgacttcacggctttgt-3′ (SEQ ID NO: 3) (SEQ ID NO: 14)(SEQ ID NO: 15) 4 CIP2A −428/+70 5′-GCTAGCagaggatgacgcacaaacgaaaaa-3′5′-CTCGAGcctctgacttcacggctttgt-3′ (SEQ ID NO: 4) (SEQ ID NO: 16)(SEQ ID NO: 17) 5 CIP2A −284/+70 5′-GCTAGCgggatctcaggccgaaaa-3′5′-CTCGAGcctctgacttcacggctttgt-3′ (SEQ ID NO: 5) (SEQ ID NO: 18)(SEQ ID NO: 19) 6 CIP2A −213/+70 5′-GCTAGCtcctggacccacaaatcacct-3′5′-CTCGAGcctctgacttcacggctttgt-3′ (SEQ ID NO: 6) (SEQ ID NO: 20)(SEQ ID NO: 21) 7 CIP2A −171/+70 5′-GCTAGCcgtcaccgagaacggtc-3′5′-CTCGAGcctctgacttcacggctttgt-3′ (SEQ ID NO: 7) (SEQ ID NO: 22)(SEQ ID NO: 23) 8 CIP2A −123/+70 5′-GCTAGCaccggatccggaagctt-3′5′-CTCGAGcctctgacttcacggctttgt-3′ (SEQ ID NO: 8) (SEQ ID NO: 24)(SEQ ID NO: 25) 9 CIP2A −95/+70 5′-GCTAGCggggtggggccgaaaatcaaa-3′5′-CTCGAGcctctgacttcacggctttgt-3′ (SEQ ID NO: 9) (SEQ ID NO: 26)(SEQ ID NO: 27) Restriction sites NheI in the forward primer and XhoI inthe reverse primer are indicated in bold.

TABLE 4 Primer Sequences Utilized For Sequencing SEQUENCE POSITION INSEQ ID NO SEQUENCE NAME pGL4.10 [luc2] SEQUENCE 28 RV primer 3 4191-42105′-ctagcaaaataggctgtccc-3′ (SEQ ID NO: 28) 29 RV primer 4 2076-20955′-gacgatagtcatgccccgcg-3 (SEQ ID NO: 29)

TABLE 5 siRNA Oligonucleotides Utilized for Ets1 and Elk1 Knock-Down SEQID siRNA Complimentary Region on Ets1 NO: Target siRNA oligonucleotideand Elk1 mRNA Accession No. 30 Ets1 CAGAAUGACUACUUUGCUACAGAATGACTACTTTGCTA (SEQ ID NO: 30) NM 001143820.1- 875-893(Variant 1)(SEQ ID NO: 34) CAGAATGACTACTTTGCTANM 005238.3- 974-992 (Variant 2) (SEQ ID NO: 35) 31 Ets1GAAAUGAUGUCUCAAGCAU GAAATGATGTCTCAAGCAT (SEQ ID NO: 31)NM 001143820.1- 344-362 (Variant 1) (SEQ ID NO: 36) GAAATGATGTCTCAAGCATNM 005238.3- 443-461 (Variant 2) (SEQ ID NO: 37) 32 Elk1GCAGCAGCCGGAACGAGUA GCAGCAGCCGGAACGAGTA (SEQ ID NO: 32)NM 001114123.1- 762-780 (Variant 1) (SEQ ID NO: 38) GCAGCAGCCGGAACGAGTANM005229.3- 656-674 (Variant 2) (SEQ ID NO: 39) 33 Elk1CGGAAGAGCUUAAUGUGGA CGGAAGAGCTTAATGTGGA (SEQ ID NO: 33)NM 001114123.1- 1014-1032 (Variant 1) (SEQ ID NO: 40)CGGAAGAGCTTAATGTGGA NM005229.3- 908-926 (Variant 2) (SEQ ID NO: 41)

TABLE 6 Primers Used in Chromatin ImmunoprecipitationREGION SPANNING CIP2A PROMOTER NUCLEOTIDE SEQUENCE−16 to −139 (within the Forward: CIP2A proximal promoter,GGACTTCCGGAGCCCGACCG which has the Ets1, Elk1 (SEQ ID NO: 44)palindromic binding Reverse: sites) CCGGCTTAGGGACCACCACCG(SEQ ID NO: 42) (SEQ ID NO: 45) −2101 to −2379 (distal Forward:region in the CIP2A AAACTGGAAATTAAAAGCGTGAGC promoter, which doesn't(SEQ ID NO: 46) have the Ets1, Elk1 Reverse: binding sites)TGCCATCTTTGTTGGATTTTGACTTA (SEQ ID NO: 43) (SEQ ID NO: 47)

TABLE 7 Primers Utilized for Construction of Ets1, Elk1Mammalian Expression Vectors SEQUENCE NO. CLONE PRIMER SEQUENCE SEQ IDEts1- Forward: ATGAGCTACTTTGTGGATTCTGCTG NO: 52 Topo (SEQ ID NO: 53)Reverse: TCACTCGTCGGCATCTGGCTTGACGTCCAG (SEQ ID NO: 54) SEQ ID Elk1-Forward: ATGGACCCATCTGTG NO: 55 Topo (SEQ ID NO: 56) Reverse:TCATGGCTTCTGGGGCCCTGGGGAGAGCAC (SEQ ID NO: 57)

What is claimed is:
 1. A pharmaceutical composition, comprising: a) afirst siRNA targeted against Ets1 mRNA, said first siRNA hybridizes to atarget sequence of said Ets1 mRNA selected from the group consisting ofSEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37; b) asecond siRNA targeted against Elk1 mRNA, said second siRNA hybridizes toa target sequence of said Elk1 mRNA selected from the group consistingof SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41; and c)a pharmaceutical acceptable carrier, wherein said pharmaceuticalcomposition inhibits gene expression of CIP2A in a cell.
 2. Thepharmaceutical composition of claim 1, wherein said first siRNA is atleast one siRNA selected from the group consisting of SEQ ID NO: 30 andSEQ ID NO:
 31. 3. The pharmaceutical composition of claim 2, whereinsaid first siRNA consists of SEQ ID NO:
 30. 4. The pharmaceuticalcomposition of claim 2, wherein said first siRNA consists of SEQ ID NO:31.
 5. The pharmaceutical composition of claim 1, wherein said secondsiRNA is at least one siRNA selected from the group consisting of SEQ IDNO: 32 and SEQ ID NO:
 33. 6. The pharmaceutical composition of claim 5,wherein said second siRNA consists of SEQ ID NO:
 32. 7. Thepharmaceutical composition of claim 5, wherein said second siRNAconsists of SEQ ID NO:
 33. 8. The pharmaceutical composition of claim 1,wherein said first siRNA contains at least one modified nucleotideselected from the group consisting of 2′-O-methyl (2′OMe),2′-deoxy-2′-fluoro (2′F), 2′-deoxy and 2′-O-(2-methoxyethyl) (MOE). 9.The pharmaceutical composition of claim 2, wherein said first siRNAcontains at least one modified nucleotide selected from the groupconsisting of 2′-O-methyl (2′OMe), 2′-deoxy-2′-fluoro (2′F), 2′-deoxyand 2′-O-(2-methoxyethyl) (MOE).
 10. The pharmaceutical composition ofclaim 1, wherein said second siRNA contains at least one modifiednucleotide selected from the group consisting of 2′-O-methyl (2′OMe),2′-deoxy-2′-fluoro (2′F), 2′-deoxy and 2′-O-(2-methoxyethyl) (MOE). 11.The pharmaceutical composition of claim 5, wherein said second siRNAcontains at least one modified nucleotide selected from the groupconsisting of 2′-O-methyl (2′OMe), 2′-deoxy-2′-fluoro (2′F), 2′-deoxyand 2′-O-(2-methoxyethyl) (MOE).
 12. The pharmaceutical composition ofclaim 1, wherein said pharmaceutical composition is suitable for oraladministration.
 13. The pharmaceutical composition of claim 1, whereinsaid pharmaceutical composition is suitable for injectionadministration.
 14. The injection administration of claim 13, whereinsaid injection is intramuscular injection.